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The SWI/SNF chromatin remodeling complex helps resolve R-loop-mediated transcription–replication conflicts

Abstract

ATP-dependent chromatin remodelers are commonly mutated in human cancer. Mammalian SWI/SNF complexes comprise three conserved multisubunit chromatin remodelers (cBAF, ncBAF and PBAF) that share the BRG1 (also known as SMARCA4) subunit responsible for the main ATPase activity. BRG1 is the most frequently mutated Snf2-like ATPase in cancer. In the present study, we have investigated the role of SWI/SNF in genome instability, a hallmark of cancer cells, given its role in transcription, DNA replication and DNA-damage repair. We show that depletion of BRG1 increases R-loops and R-loop-dependent DNA breaks, as well as transcription–replication (T-R) conflicts. BRG1 colocalizes with R-loops and replication fork blocks, as determined by FANCD2 foci, with BRG1 depletion being epistatic to FANCD2 silencing. Our study, extended to other components of SWI/SNF, uncovers a key role of the SWI/SNF complex, in particular cBAF, in helping resolve R-loop-mediated T-R conflicts, thus, unveiling a new mechanism by which chromatin remodeling protects genome integrity.

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Fig. 1: Analysis of DNA damage and genome instability in siBRG1 cells.
Fig. 2: Effect of BRG1 depletion on DNA replication dynamics.
Fig. 3: Genome-wide analysis of R-loop accumulation on BRG1 depletion.
Fig. 4: Epistatic analysis between BRG1 and known R-loop-preventing factors.
Fig. 5: Evaluation of BRG1 occurrence at replication fork-stalling sites.
Fig. 6: BRG1 genome-wide colocalization analysis with R-loop, replication fork stalling and DNA-damage sites.
Fig. 7: Chromatin accessibility and nucleosome occupancy analysis at R-loop-gain sites in BRG1-deficient cells.
Fig. 8: R-loop and DNA-damage study in cells depleted of different SWI/SNF subunits.

Data availability

The siBRG1 DRIPc-seq and RNA-seq datasets, the first siC DRIPc-seq replicate and one DRIP-seq replicate dataset have been deposited at the Gene Expression Omnibus repository and are available under accession no. GSE154631. Original data for another DRIP-seq replicate, siC RNA-seq and the second siC DRIPc-seq replicate dataset are available at the same database under accession no. GSE127979 (ref. 14), even though all the experiments were performed in parallel. Other publicly available genome-wide data used in the present study are listed in Supplementary Table 1. Cancer-related information for SWI/SNF genes was retrieved from cBio Cancer Genomics Portal (www.cbioportal.org)35,107 and Integrative Onco Genomics (www.intogen.org)108,109 databases. Source data are provided with this paper.

Code availability

Software and algorithms source and links are listed in Supplementary Table 1.

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Acknowledgements

We thank J. C. Reyes (CABIMER, Seville) for reagents provided and discussions, and the Genomic Unit of CABIMER for DNA sequencing. Research was funded by grants from the European Research Council (grant no. ERC2014 AdG669898 TARLOOP), the Spanish Ministries of Economy and Competitiveness (grant no. BFU2016-75058-P) and Science and Innovation (grant no. PDI2019-104270GB-I00/BMC), the European Union (FEDER) and Foundation ‘Vencer el Cancer’. A.B-F. was supported in part by a Juan de la Cierva postdoctoral contract (FJCI-2017-34536) from the Spanish Ministry of Science and Innovation.

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A.B.-F. and A.A. designed the study and the experiments. A.B.-F. performed most of the experiments and all the bioinformatic analysis. S.B. and S.M. contributed with specific experiments. A.B.-F and A.A. wrote the manuscript. All authors read, discussed and agreed with the final version of this manuscript.

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Correspondence to Andrés Aguilera.

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The authors declare no competing interests.

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Peer review information Nature Genetics thanks Stephan Hamperl and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Analysis of DNA damage and genome instability in siBRG1 cells.

a, Quantification of nuclear BRG1 signal intensity in control (siC) and BRG1-depleted (siBRG1) HeLa cells. Data plotted as scatter plot (n = 3). Scale bar, 20 μm. (Mann-Whitney U test, two-tailed). b, WB of chromatin fraction from cells treated as in (a) using anti-BRG1 antibody (n = 2). Pounceau S membrane staining is shown as loading control. c, Quantification of depletion efficiency in cells treated as in (a) as measured by cDNA. Data are plotted as mean + SEM (n = 3). (Unpaired Student’s t-test, two-tailed). d, Representative images of γH2AX IF in control (siC) and BRG1-depleted (siBRG1) cells with (+) and without (−) RNH1 overexpression. Scale bar, 5 μm. e, Quantification of nuclear S9.6 signal intensity in control cells (siC) and BRG1-depleted cells using either a pool or individual siRNAs. Data presented as scatter plot (n = 3). Scale bar, 10 μm. (Mann-Whitney U test, two-tailed). f, Quantification of S9.6 foci per cell (left) and nucleolar signal intensity (right) in cells treated as in (d). Data plotted as scatter plot (n = 3). Representative images are shown. Scale bar, 10 μm. (Mann-Whitney U test, two-tailed). g, DRIP-qPCR expressed as % of input in cells treated as in (a). Data plotted as mean + SEM (n = 3). (Paired Student’s t-test, one-tailed). h, Quantification of nuclear S9.6 signal intensity in control cells (siC) and BRG1-depleted (siBRG1) cells transfected either with an empty plasmid (−) or a plasmid expressing an siRNA-resistant wild-type (WT) or catalytically-dead (K785R) BRG1. Data presented as scatter plot (n = 3). Scale bar, 10 μm. (Mann-Whitney U test, two-tailed). i, Percentage of cells in each cell cycle phase in cells treated as in (d). Cell cycle profiles (left) and percentages of cells for each cell cycle phase plotted as mean + SD (right) are shown (n = 3). (Paired Student’s t-test, two-tailed). P-values are indicated. Other details as in Fig. 1.

Source data

Extended Data Fig. 2 Effect of BRG1 depletion on DNA replication dynamics.

a, Quantification of nuclear EdU signal intensity in EdU+ control (siC) and BRG1-depleted (siBRG1) HeLa cells with (+) and without (−) RNH1 overexpression. Data plotted as box plot (n = 3). Scale bar, 10 μm. (Mann-Whitney U test, two-tailed). b, Representative FANCD2 IF (red) examples from cells treated as in (a) (n = 3). Scale bar, 5 μm. c, Representative BLM IF (green) examples from siC and siBRG1-treated cells (n = 2). Scale bar, 5 μm. d, Percentage of EdU-positive cells showing FANCD2 foci in siBRG1 cells. Scale bar, 10 μm. Data plotted as mean + SEM (n = 3). e, Percentage of EdU-positive cells showing BLM foci in siBRG1 cells. Scale bar, 10 μm. Data are plotted as mean + SEM (n = 3). f, Quantification of DNA (DAPI;blue) and FANCD2 (red) profiles along the indicated cell slices (yellow line) in siBRG1 cells. Scale bar, 5 μm. g, Quantification of DNA (DAPI;blue) and BLM (green) profiles along the indicated cell slices (yellow line) in siBRG1 cells. Scale bar, 5 μm. h, Representative S9.6 IF images in HeLa cells treated as in (c) along the cell cycle (n = 3). Cell phases are indicated (G1 (dark blue), S (light blue) and G2 (green)). Scale bar, 10 μm. i, Quantification of S9.6 intensity by flow cytometry in cells treated as in (c) along the cell cycle. Fold changes on S9.6 signal from G1 to S+G2/M are plotted as scatter plot (n = 3). Mean + SD are shown. (Paired Student’s t-test, one-tailed). j, Quantification of chromatin-bound BRG1 signal intensity along the cell cycle. Data plotted as box plot (n = 3). Representative images are shown. Cell cycle phases are indicated as in (h). Scale bar, 10 μm. (Mann-Whitney U test, two-tailed). k, Representative BRG1+PCNA PLA images in wild-type HeLa cells. Controls and positive PLA (red) are shown (n = 1). Scale bar, 10 μm. P-values are indicated. Other details as in Figs. 1 and 2.

Source data

Extended Data Fig. 3 Genome-wide analysis of R-loop accumulation upon BRG1 depletion.

a, DRIP-seq mean coverage over gene bodies (±2kb) from control samples (n = 2). b, DRIP-qPCR in control K562 cells. Immunoprecipitation values normalized to untreated samples. Data are mean + SEM (n = 3). (Paired Student’s t-test, one-tailed). c, DRIP-seq mean-coverage over DRIPc-seq peaks (n = 2). d, Length of siC DRIPc-seq peaks (n = 2). e, Genome annotation of DRIPc peaks of siC and R loop-gain peaks siBRG1 K562 cells. f, Vulcano plot of DRIPc-seq differential analysis between siC (blue) and siBRG1 (vermillion) K562 cells (n = 2). Dots represent peaks. g, Absolute numbers of de novo and increasing DRIPc-seq peaks of siBRG1 cells (n = 2). h, Representative screenshot comparing siC and siBRG1 DRIPc-seq profiles considering or discarding Alu-containing reads. Watson-Crick strand reads shown together (n = 2). i, Metagenomic analysis of DRIPc-seq mean coverages along gene bodies (n = 2). j, BRG1 ChIP-seq average coverage (n = 2) over genes presenting R-loop-gain peaks distributed in quartiles according to the R loop-gaining rate of DRIPc-seq. Dots represent genes. Regression line in red. k, Venn diagram with BRG1-bound and R-loop-gain genes after siBRG1 (Hypergeometric test). l, Gene lengths of entire genome and R-loop-gain genes (Mann-Whitney U-test, two-tailed). m, Expression of R-loop-gain genes in siC and BRG1-depleted cells (Mann-Whitney U-test, two-tailed). n, Box-plot of GC-content. Details as in (l). o, Metaplot of GC-skew along gene body over gene sets of (l). Dark and light-colored lines represent genes from each strand. p, Box-plot of expression values. Details as in (l). q, Metaplot of DRIPc-seq mean coverage increase (ΔDRIPc-seq) upon BRG1-depletion over chromosomes or rDNA (n = 2). r, Venn diagram showing R-loop-forming genes in siC cells and R-loop-gain genes in siBRG1 and siUAP56 K562 cells. Other details as in Figs. 13.

Source data

Extended Data Fig. 4 Epistatic study between BRG1 and known R loop-preventing factors.

a, Depletion controls by WB in double-knockdown experiments. siRNAs and antibodies used are indicated (n = 2). b, Representative S9.6 IF in HeLa cells transfected with the indicated siRNAs combined either with siC (−) or siBRG1 (+) (n = 3; except for siC, siTHOC1, siTHOC1+siBRG1, siUAP56 and siUAP56+siBRG1 where n = 4). Scale bar, 5 μm. c, Representative γH2AX IF in HeLa cells treated as in (b) (n = 3; except for siC and siFANCD2 where n = 4). Scale bar, 10 μm. d, Quantification of nuclear S9.6 signal intensity through cell cycle in siTHOC1 single depletion and siTHOC1+siBRG1 double depleted HeLa cells. Data presented as box plot (n = 3). Scale bar, 10 μm. (Mann-Whitney U test, two-tailed). e, Quantification of nuclear S9.6 signal intensity through cell cycle in siUAP56 single depletion and siUAP56+siBRG1 double depleted HeLa cells. Data presented as box plot (n = 3). Scale bar, 10 μm. (Mann-Whitney U test, two-tailed). f, Representative RNAPII S2P+PCNA PLA example images in wild-type HeLa cells. Control PLAs are also shown (n = 1). Scale bar, 10 μm. g, Quantification of RNAPII S2P+PCNA PLA in control cells (siC) and BRG1-depleted cells (siBRG1) either transfected with an empty plasmid (−) or a plasmid allowing expression of a siRNA-resistant wild-type (WT) or catalytically dead (K785R) version of BRG1. Data presented as scatter plot (n = 3). Scale bar, 10 μm. (Mann-Whitney U test, two-tailed). h, Representative RNAPII S2P+PCNA PLA example images in HeLa cells treated as in (b) (n = 3; except for siC (n = 6) and siSETX (n = 4)). Scale bar, 10 μm. P-values are indicated. Other details as in Figs. 1 and 2.

Source data

Extended Data Fig. 5 Evaluation of BRG1 occurrence at RF stalling sites.

a, Representative FANCD2+BRG1 PLA example images in wild-type HeLa cells. Control PLAs are also shown (n = 1). b, Representative FANCD2+BRG1 PLA example images in control (siC) and UAP56-depleted (siUAP56) HeLa cells with (+) and without (−) overexpression of RNH1. (n = 3). c, Representative RPA S4/8P+BRG1 PLA example images in wild-type HeLa cells. Control PLAs are also shown (n = 1). d, Representative γH2AX+BRG1 PLA example images in wild-type HeLa cells (n = 1). e, Representative RPA S4/8P+BRG1 PLA example images in siC, siUAP56 and siUAP56+siBRG1-transfected HeLa cells (n = 3). f, Representative γH2AX+BRG1 PLA example images in HeLa cells treated as in (e) (n = 3). g, Representative S9.6+BRG1 PLA example images in wild-type HeLa cells (n = 1). h, Representative S9.6+BRG1 PLA example images in HeLa cells treated as in (b) (n = 3). Scale bar, 10 μm. Other details as in Fig. 1.

Extended Data Fig. 6 BRG1 genome-wide co-localization analysis with R loop, RF stalling and DNA damage sites.

a, Venn diagram showing BRG1 (yellow), FANCD2 (red) and γH2AX (blue) target genes co-occurrence in control K562 cells. b, DRIPc-seq (green), BRG1 (yellow), FANCD2 (red) and γH2AX (purple) ChIP-seq data mean coverage around T-R conflicts (±1Mb). Data presented as metaplot according to RF direction. c, Quantification of co-directional (CD; green bar) vs head-on (HO; red bar) T-R conflicts. Absolute numbers are plotted. d, BRG1 (yellow) and FANCD2 (red) ChIP-seq data mean coverage around HO T-R conflicts (±1Mb). Data presented as metaplot according to RF direction. e, As in (d), but around CD T-R conflicts (±1Mb). f, DRIPc-seq average coverage at DRIPc-seq peaks reached by the RF in CD or HO orientation during DNA replication. Data plotted as box plot. (Mann-Whitney U test, two-tailed). g, BRG1 ChIP-seq average coverage at genes reached by the RF in HO and CD orientation. Data plotted as box plot. (Mann-Whitney U test, two-tailed). h, FANCD2 ChIP-seq average coverage at genes reached by the RF in HO and CD orientation. Data plotted as box plot. (Mann-Whitney U test, two-tailed). P-values are indicated. Other details as in Figs. 2 and 6. Average coverages from two DRIPc-seq and BRG1 ChIP-seq biological replicates are shown.

Source data

Extended Data Fig. 7 Chromatin accessibility and nucleosome occupancy analysis at R loop gain sites in BRG1-deficient cells.

a, Correlation analysis between K562 and HAP1 BRG1 ChIP-seq analysis. b, Correlation analysis between K562 and CD36 BRG1 ChIP-seq analysis. c, Correlation analysis between BRG1 ChIP-seq in K562 and BIN67 (BRG1-deficient) cells expressing BRG1. d, Representative γH2AX IF example images in HeLa cells transfected with the indicated siRNAs and either en empty plasmid (−) or a plasmid for RNH1 overexpression (+). Scale bar, 10 μm. e, Representative S9.6 IF example images in HeLa cells transfected with the indicated siRNAs and treated as in (d). Scale bar, 10 μm. f, Representative screenshot comparing siC (blue) and siBRG1 (vermillion) DRIPc-seq data in K562 and ChIP-seq profiles of BRG1 (yellow), BRM (green), ARID1A (orange) and PBRM1 (purple) in HAP1 cells. g, BRG1, BRM, ARID1A and PBRM1 ChIP-seq mean coverage in HAP1 WT cells over genes identified as R loop-gain genes in siBRG1 K562 (± 2kb). Data plotted as metagene. h, BRG1, BRM, ARID1A and PBRM1 ChIP-seq mean coverage in HAP1 WT cells around R loop accumulating peaks in siBRG1 K562 cells (± 10kb). i, HAP1 WT, BRG1 KO, BRM KO, ARID1A KO and PBRM1 KO ATAC-seq average coverage at R loop gain sites identified in siBRG1 K562 cells. Data plotted as box plot. (Mann-Whitney U test, two-tailed). j, HAP1 WT, BRG1 KO, BRM KO, ARID1A KO and PBRM1 KO ATAC-seq mean coverage around HO (left) and CD (right) T-R collisions (± 1Mb). Data plotted as heatmap according to RF direction. Pearson correlations (r) are shown in (a), (b), and (c). P-values are indicated in (i). Other details as in Figs. 2 and 6.

Source data

Extended Data Fig. 8 SWI/SNF mutation and cancer.

a, Number of identified mutations in cancer from a curated set on non-redundant cancer studies (www.cbioportal.com) for the main SWI/SNF (red), ISWI (yellow), CHD (blue) and INO80 (green) ATPase subunits. b, Number of cancer types where the gene is considered a cancer driver gene (www.intogen.org) for the main SWI/SNF (red), ISWI (yellow), CHD (blue) and INO80 (green) ATPase subunits. c, Distribution of cancer-associated BRG1 mutations along the protein. Protein length (grey) and domains (colored squares) are drawn. Highly frequently and recurrently altered in cancer protein residues (cancer hotspots) are highlighted as orange dots in the upper track. K785R point mutation used in rescue experiments is indicated. d, Quantification of nuclear S9.6 signal intensity in HeLa (BRG1-proficient) and C-33 A (BRG1-deficient) cells. Data presented as scatter plot (n = 3). Scale bar, 10 μm. (Mann-Whitney U test, two-tailed). e, Number of identified mutations in cancer from a curated set on non-redundant cancer studies (www.cbioportal.com) for the main SWI/SNF genes. f, Number of cancer types where the gene is considered a cancer driver gene (www.intogen.org) for the main SWI/SNF genes. P-values are indicated. Other details as in Fig. 1.

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Bayona-Feliu, A., Barroso, S., Muñoz, S. et al. The SWI/SNF chromatin remodeling complex helps resolve R-loop-mediated transcription–replication conflicts. Nat Genet (2021). https://doi.org/10.1038/s41588-021-00867-2

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